Angiogenesis is the process of new growth from pre-existing blood vessels during development, in normal physiology, and numerous pathologies. Heterogeneity, in both the molecular identity of vascular endothelial cells (EC) and the factors that act on them, is an essential aspect of normal vascular development. Notch signaling regulates EC heterogeneity, permitting or repressing specific signaling pathways within individual cells. Additional signaling complexity is achieved by the actions of non-VEGF-A signaling pathways, including the bone morphogenetic protein (BMP) pathway. These complex interactions promote angiogenesis suited to specific tissue micro-environments. BMP2 is a context-dependent pro-angiogenic ligand; necessary for venous angiogenesis in the developing zebrafish, yet dispensable for arterial angiogenesis. In mammalian systems, BMP signaling promotes branching of angiogenic vessels in several models. Notch is a well-established inhibitor of VEGF-A signaling, and has only recently been shown to intersect with the BMP pathway in EC. However, studies of Notch-BMP interactions in EC have largely focused on the synergistic effects of the two pathways on Notch target gene expression, and have not explored the possibility that Notch regulates more upstream aspects of BMP signal transduction, such as phosphorylation of receptor-associated SMADs (R-SMADs). My preliminary results imply Notch negatively regulates BMP2- or BMP6-dependent R-SMAD phosphorylation. Therefore, I hypothesize that Notch negatively regulates an upstream component of BMP signaling in EC, thereby contributing to the endothelial cell heterogeneity necessary for proper vascular patterning. The proposed study will rigorously test this hypothesis in vivo and in vitro in 3 Aims. Aim 1 will determine if Notch activity regulates the ERK1/2 cascade downstream of BMP ligand binding. Aim 2 will determine if the inhibitory SMAD, SMAD6 is a bona fide Notch target and mediates Notch-dependent BMP pathway regulation. Aim 3 will utilize live-imaging techniques to monitor the effect of Notch-BMP pathway integration on angiogenesis in real-time in vitro. This proposal is both conceptually and technically innovative. Current dogma assumes Notch and BMP pathway co- activation in EC results in feedback inhibition of BMP signaling and EC migration. However, my hypothesis is predicated on the novel finding that Notch activation prior to BMP ligand engagement in an EC renders it unresponsive to BMP ligands. This proposal will validate my hypothesis and reveal a previously unknown mechanism of Notch-dependent regulation of angiogenesis. One technical innovation in this proposal is to translate the promising CRISPRi RNA-guided gene knockdown technology to zebrafish. This innovation has the possibility to radically transform genetic studies in zebrafish and will be made available to the entire scientific community. A second innovation is the real-time observation of Notch-BMP pathway integration and the subsequent consequences to single-cell phenotypes in sprouting angiogenesis.